1. Field of the Invention
The present invention relates to a soluble Fc receptor and a method for its production.
2. Description of the Background Art
Immunoglobulins, also referred to as antibodies, are a major component of the humoral immune response of all mammals. These glycoproteins are divided into five major structural classes, each of which can be divided into subclasses. The five major classes are IgA, IgD, IgE, IgG, and IgM. IgG is the most common class of immunoglobulins found in the serum.
It is the highly specific interaction between an antibody and its target antigen that makes an immunoglobulin an effective agent against an invading pathogen. The high specificity of the immunoglobulin--antigen interaction allows the immune system to attack the foreign antigen with minimal harm to the host organism.degree. In general, immunoglobulins have a high affinity for their target antigen with an average dissociation constant (Kd) for the antigen--antibody complex on the order of 10.sup.-9 to 10.sup.-12 moles/liter.
Immunoglobulins are composed of four polypeptide chains; two identical light chains, each of approximately 25 kD in molecular mass and two identical heavy chains, each of approximately 50-80 kD in molecular mass. The different classes of antibodies are distinguished by structural differences in their heavy chains. A schematic drawing of an IgG 10 is shown in FIG. 1.
Light chains 12 and 14 have been characterized as having a single amino terminal variable domain (a domain is a distinct region of tertiary protein structure), V.sub.L and a single constant domain, C.sub.L. The term variable refers to the variability in amino acid sequence found in this type of domain between antibodies with different antigen binding specificities, which antibodies are of the same immunoglobulin class and subclass. The heavy chains 16 and 18 of an IgG contain a single amino terminal variable domain, VH, followed by three constant domains, C.sub.H 1, C.sub.H 2, and C.sub.H 3, as shown in FIG. 1.
In general, an immunoglobulin, and in particular IgG, may be characterized as a Y shaped molecule in which each upper arm of the Y is formed by a pairing of a single light chain (V.sub.L+ C.sub.L) with the two most amino terminal domains of a single heavy chain (V.sub.H+ C.sub.H1). It is the pairing of the variable domains of the light and heavy chains that forms the antigen binding sites 20 and 22. The constant domain of the light chain (C.sub.L) interacts with the first constant domain of the heavy chain .(C.sub.H 1). The heavy and light chains are covalently bound to each other by a disulfide bond 24 between the paired C.sub.L and C.sub.H 1 domains. The two heavy chains dimerize through interactions between their 2nd and 3rd constant domains (C.sub.H 2 and C.sub.H 3). The two heavy chains are also covalently bound to each other through interchain disulfide bonds 26. These disulfide bonds 26 connect the two heavy chains in a region between the C.sub.H 1 and C.sub.H 2 domains that is known as the hinge region. Beneath the hinge region are oligosaccharide units (carbohydrates) 28 and 30, which are attached to the C.sub.H 2 domains within the structure. The V.sub.L and C.sub.L of the light chain paired with V.sub.H and C.sub.H 1 of the heavy chain is called an Fab, FIG. 1 at 32 and 34; and, the paired C.sub.H 2 and C.sub.H 3 domains of the IgG heavy chains is referred to as the Fc, FIG. 1 at 36, of the antibody.
Typically, immunoglobulin molecules are soluble in aqueous solution and bind to compounds in a highly specific manner with a great affinity. It is possible to produce antibodies against virtually any organic compound known to humans. These characteristics of immunoglobulins have resulted in their widespread analytical and therapeutic use throughout the biological sciences.
Fc receptor is a general term that refers to any one of several proteins that bind to the Fc region of an immunoglobulin. Fc receptors can be soluble or membrane-bound. An example of a membrane-bound Fc receptor is the FcRn from the intestines of neonatal rats. The FcRn was recently cloned and characterized by N. E. Simister and K. E. Mostov, as described in "An Fc Receptor Structurally Related to MHC Class I Antigens" Nature (London) Vol. 337, pp. 184-187 (1989).
__________________________________________________________________________ Met Gly Met Ser Gln Pro Gly Val Leu Leu Ser Leu Leu Leu Val Leu -22 -20 -15 -10 Leu Pro Gln Thr Trp Gly Ala Glu Pro Arg Leu Pro leu Met Tyr His -5 -1 +1 5 10 Leu Ala Ala Val Ser Asp Leu Ser Thr Gly Leu Pro Ser Phe Trp Ala 15 20 25 Thr Gly Trp Leu Gly Ala Gln Gln Tyr Leu Thr Tyr Asn Asn Leu Arg 30 35 40 Gln Glu Ala Asp Pro Cys Gly Ala Trp Ile Trp Glu Asn Gln Val Ser 45 50 55 Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Lys Ser Lys Glu Gln Leu 60 65 70 Phe Leu Glu Ala Ile Arg Thr Leu Glu Asn Gln Ile Asn Gly Thr Phe 75 80 85 90 Thr Leu Gln Gly Leu Leu Gly Cys Glu Leu Ala Pro Asp asn Ser Ser 95 100 105 Leu Pro Thr Ala Val Phe Ala Leu Asn Gly Glu Glu Phe Met Arg Phe 110 115 120 Asn Pro Arg Thr Gly Asn Trp Ser Gly Glu Trp Pro Glu Thr Asp Ile 125 130 135 Val Gly Asn Leu Trp Met Lys Gln Pro Glu Ala Ala Arg Lys Glu Ser 140 145 150 Glu Phe Leu Leu Thr Ser Cys Pro Glu Arg Leu Leu Gly His Leu Glu 155 160 165 170 Arg Gly Arg Gln Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu 175 180 185 Lys Ala Arg Pro Gly asn Ser Gly Ser Ser Val Leu Thr Cys Ala Ala 190 195 200 Phe Ser Phe Tyr Pro Pro Glu Leu Lys Phe Arg Phe Leu Arg Asn Gly 205 210 215 Leu Ala Ser Gly Ser Gly Asn Cys Ser Thr Gly Pro Asn Gly Asp Gly 220 225 230 Ser Phe His Ala Trp Ser Leu Leu Glu Val Lys Arg Gly Asp Glu His 235 240 245 250 His Tyr Gln Cys Gln Val Glu His Glu Gly Leu Ala Gln Pro Leu Thr 255 260 265 Val Asp Leu Asp Ser Pro Ala Arg Ser Ser Val Pro Val Val Gly Ile 270 275 280 Ile Leu Gly Leu Leu Leu Val Val Val Ala Ile Ala Gly Gly Val Leu 285 290 295 Leu Trp Asn Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Leu Ser Leu 300 305 310 Ser Gly Asp Asp Ser Gly Asp Leu Leu Pro Gly Gly Asn Leu Pro Pro 315 320 325 330 Glu Ala Glu Pro Gln Gly Val Asn Ala Phe Pro Ala Thr Ser. 335 340 344 __________________________________________________________________________
This Fc receptor (FcRn) is physiologically expressed on the luminal surface of neonatal rat intestinal epithelial cells. The FcRn was determined to optimally bind to IgG at the intestinal pH of 6-6.5 and to release bound IgG at the serosal pH of approximately 7.5. The physiological role of FcRn is to bind to maternal IgG consumed by the newborn when it drinks its mother's milk. The FcRn is then involved in the transport of the bound IgG across the intestinal epithelial barrier and the release of the IgG into the blood of the newborn. By this means the neonatal rat can passively acquire some resistance to disease. This is especially important during the first few weeks of independent life of the rat (as well as the cat) because at birth these mammals are practically agammaglobulinemic (without antibodies). FcRn, as found on the surface of neonatal rat intestinal epithelial cells, is a heterodimer consisting of an FcRn heavy chain (p51) of approximately 45-53 kD in molecular mass and a light chain (.beta..sub.2 m) of approximately 14 kD in molecular mass. The FcRn heavy chain appears to have 3 extracellular domains, a transmembrane domain and a cytoplasmic tail. The three extracellular domains of the FcRn heavy chain have significant sequence similarity to the corresponding domains of Class I Major Histocompatibility Complex (MHC) molecules. This sequence similarity suggests that the FcRn may have a tertiary protein structure similar to that observed for Class I MHC molecules. The FcRn light chain is a .beta..sub.2 m (.beta.-2 microglobulin), a soluble single domain protein also found as a component of the Class I MHC molecule heterodimer.
The sequence for rat .beta..sub.2 m was published by J. Sundelin et al. Scand. J. Immunol. 27, pp. 195-199 (1988) as follows:
__________________________________________________________________________ Ile Gln Lys Thr Pro Gln Ile Gln Val Tyr Ser Arg His Pro Pro Glu 1 5 10 15 Asn Gly Lys Pro Asn Phe Leu Asn Cys Tyr Val Ser Gln Phe His Pro 20 25 30 Pro Gln Ile Glu Ile Glu Leu Leu Lys Asn Gly Lys Lys Ile Pro Asn 35 40 45 Ile Glu Met Ser Asp Leu Ser Phe Ser Lys asp Trp Ser Phe Tyr Ile 50 55 60 Leu Ala His Thr Glu Phe Thr Pro Thr Glu Thr Asp Val Tyr Ala Cys 65 70 75 80 Arg Val Lys His Val Thr Lys Leu Glu Pro Lys Thr Val Thr Trp Asp 85 90 95 Arg Asp Met. 99 __________________________________________________________________________
The transmembrane domain of the FcRn heavy chain anchors the FcRn heterodimer into the cell membrane of the intestinal epithelial cells. The hydrophobic nature of this transmembrane domain precludes the solubilization of this protein in an aqueous buffer in the absence of surfactants, which are often toxic, which can reduce the stability of proteins, and which are often difficult to remove once they have been in contact with the protein.
There are numerous potential applications for an Fc receptor which is soluble in aqueous solutions without the use of a surfactant. In addition, an Fc receptor, such as the FcRn, which is capable of binding antibodies at one pH and releasing the antibodies at another, is one that could be used in the biotechnology industry. Fc receptors can be used in medicine for the detection of antibodies under conditions of physiological pH. In addition, in U.S. patent application, Ser. No. 07/819,040, of Andrew Huber et al., filed Jan. 10, 1992, it is disclosed that soluble FcRn receptors of the kind disclosed and claimed herein can be attached to a surface and used to concentrate and purify antibodies from mixtures comprising antibodies such as IgG from blood, ascites or tissue culture supernatants (growth medium in which cells are cultured).
However, the usefulness of the membrane-bound FcRn is limited by the fact that, like other transmembrane proteins, it is not readily soluble in an aqueous solution in the absence of surfactants, as described above.
Although cDNA for the membrane-bound FcRn was available from N. E. Simister, it was questionable whether a soluble FcRn could be produced. The soluble FcRn, like other proteins must be able to fold into a functional three dimensional structure. This folding of a protein in solution is a very complex process and is influenced by many factors, such as the presence of other proteins which assist in the folding process. Upon removal of the transmembrane domain from the FcRn, the resultant protein might not be able to fold properly in solution, rendering it non-functional and probably insoluble. Even if the protein were able to fold properly, it might not be secreted from the cell in which it is produced and/or it might simply be degraded within the cell. Further, depending on where the heavy chain of FcRn is truncated to remove the transmembrane domain, the truncated heavy chain might be less heat stable and might not associate with its intended light chain, which would affect its ability to bind to an antibody.
Definitions
The following definitions are for use in understanding the descriptions presented throughout the present application.
FcRn is understood to mean rat neonatal, intestinal, pH-determinable membrane-bound Fc receptor(s); PA1 pHsFcRn is understood to mean a soluble form of FcRn produced by applicants; PA1 pHFcR is understood to mean a membrane-bound fc receptor having the ability to bind to an antibody monomer or complex thereof at one pH and to release the antibody or complex thereof at another pH; PA1 sFcR is understood to mean soluble, vertebrated-derived Fc receptor in general; PA1 pHsFcR is understood to mean soluble, vertebrate-derived Fc receptor having the ability to bind to an antibody monomer or complex thereof at one pH and to release the antibody or complex thereof at another pH; PA1 .alpha.MEM is understood to mean a commercially available growth medium supplied by Irvine Scientific and Gibco/BRL; PA1 .beta..sub.2 m is understood to mean .beta..sub.2 -microglobulin; PA1 cDNA is understood to mean complementary DNA prepared from mRNA. PA1 DAF is understood to mean decay-accelerating factor; PA1 DMEM is understood to mean a commercially available growth medium supplied by Irvine Scientific and Gibco/BRL; PA1 MHC is understood to mean major histocompatibility complex; PA1 MSX is understood to mean L-methionine sulfoximine; and PA1 PI-PLC is understood to mean phosphatidylinositol--specific phospholipase C; PA1 (a) creating a gene fragment for a truncated heavy chain of the desired vertebrate-derived pHsFcR, said fragment excluding the transmembrane domain of said heavy chain; PA1 (b) forming an expression vector comprising a gene useful as a selectable marker and optionally a means of gene amplification, the pHsFcR heavy chain gene fragment from step (a), and a gene fragment for the light chain compatible with the pHsFcR heavy chain gene fragment of step (a); PA1 (c) introducing the expression vector of step (b) into a procaryotic or eucaryotic cell; PA1 (d) selecting for a procaryotic or eucaryotic cell comprising the modified and optionally amplified cDNAs of the desired vertebrate-derived pHsFcR; PA1 (e) growing the cell of step (d), from which the desired pHsFcR is harvested. PA1 (a) creating a gene fragment comprising a vertebrate-derived Fc receptor truncated heavy chain, the fragment excluding the transmembrane domain of the heavy chain but including a signal specifying the attachment of a lipid; PA1 (b) forming an expression vector comprising a gene useful as a selectable marker and optionally a means of gene amplification, the step (a) heavy chain gene fragment including the lipid attachment signal, and a gene fragment for the light chain compatible with the pHFcR heavy chain gene fragment of step (a); PA1 (c) introducing the expression vector of step (b) into a procaryotic or eucaryotic cell; PA1 (d) selecting a procaryotic or eucaryotic cell comprising the modified and optionally amplified cDNA of the desired vertebrate-derived pHFcR; PA1 (e) growing the cell of step (d) from which the desired pHFcR is obtained; PA1 (f) cleaving the pHFcR lipid linkage of the step (d) cell membrane to produce a pHsFcR.